WO1994008759A1 - Procede et appareil d'execution robotique de reactions de sequencage de didesoxynucleotides de sanger - Google Patents

Procede et appareil d'execution robotique de reactions de sequencage de didesoxynucleotides de sanger Download PDF

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Publication number
WO1994008759A1
WO1994008759A1 PCT/US1993/009961 US9309961W WO9408759A1 WO 1994008759 A1 WO1994008759 A1 WO 1994008759A1 US 9309961 W US9309961 W US 9309961W WO 9408759 A1 WO9408759 A1 WO 9408759A1
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Prior art keywords
microtiter plate
hand
robot
robotic system
pipetting
Prior art date
Application number
PCT/US1993/009961
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English (en)
Inventor
James Joseph Earley
Gerardus Cornelus Tromp
Darwin Johnson Prockop
Sisko Helena Kuivaniemi
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Thomas Jefferson University
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Publication of WO1994008759A1 publication Critical patent/WO1994008759A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0099—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor comprising robots or similar manipulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/028—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having reaction cells in the form of microtitration plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1002—Reagent dispensers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00353—Pumps
    • B01J2219/00355—Pumps peristaltic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00364—Pipettes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00495—Means for heating or cooling the reaction vessels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/0059—Sequential processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00689—Automatic using computers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00691—Automatic using robots
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00695—Synthesis control routines, e.g. using computer programs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00346—Heating or cooling arrangements
    • G01N2035/00445—Other cooling arrangements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00346—Heating or cooling arrangements
    • G01N2035/00455—Controlling humidity in analyser
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0401—Sample carriers, cuvettes or reaction vessels
    • G01N2035/0418—Plate elements with several rows of samples
    • G01N2035/042—Plate elements with several rows of samples moved independently, e.g. by fork manipulator
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0401—Sample carriers, cuvettes or reaction vessels
    • G01N2035/0418—Plate elements with several rows of samples
    • G01N2035/0425—Stacks, magazines or elevators for plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
    • G01N35/1011—Control of the position or alignment of the transfer device
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1065—Multiple transfer devices

Definitions

  • the present invention relates to the robotic execution of reactions to determine the DNA sequence of template DNA and more particularly to the robotic execution of Sanger dideoxynucleotide sequencing reactions.
  • DNA deoxyribonucleic acid
  • the genetic code is dependent on the chemical structure of the DNA. As described below, DNA is composed of four building blocks that are chemically bonded to one another to form long polymeric strands. Although DNA is composed of only four building blocks, successive addition of building blocks rapidly results in a structure with a unique sequence or combination of building blocks. The possible number of combinations formed by successive additions of building blocks rapidly becomes very large as it is the number of blocks to the fourth power. Determination of the sequence of an organism's DNA and therefore determination of the sequence of the genes of that organism is essential to the understanding of biology and genetic disease.
  • the DNA of all higher living organisms exists in a double-stranded form that is composed of two complementary, anti-parallel polymeric chains, also called strands (hence double-stranded) .
  • Each of the chains is in turn composed of four building blocks called deoxynucleosides.
  • Each deoxynucleoside is composed of a purine or pyrimidine ring structure called a base, a sugar moiety called deoxyribose and a phosphate group.
  • the four deoxynucleosides are adenosine (A) , cytosine (C) , guanine (G) and thymidine (T) .
  • Deoxynucleosides are covalently attached to each other by phosphodiester bonds between the deoxyribose and the phosphate moieties to form the sugar-phosphate backbone of the strand. Because no bonds of the bases are a part of the structure of the backbone of the strand, the bases can be viewed as side-chains of the sugar-phosphate backbone.
  • Each backbone has an orientation because the deoxyribose is an asymmetric molecule and the phosphodiester bonds are formed at hydroxyl groups at the number 3 and number 5 carbons. Conventionally, orientation is designated as 5' to 3' or 3' to 5'. The orientations of the two backbones of the double-stranded DNA molecule are opposite to each other and the strands are said to be anti-parallel.
  • the genetic information in DNA is encoded by the order or sequence in which the four deoxynucleosides are arranged in the polymeric chains.
  • the order of deoxynucleosides in one chain determines the order of the deoxynucleosides in the opposite chain because the bases specifically interact with bases on the opposite strand to form stable complementary pairs of bases such that A pairs with T and C pairs with G. Because of the base-pairing it is possible to determine the sequence of one strand and infer the sequence of the complementary, anti-parallel strand.
  • DNA is synthesized in cells from building blocks that are deoxyribonucleotides.
  • deoxyribonucleosides They are similar to deoxyribonucleosides but have a triphosphate moiety instead of the single phosphate found in deoxynucleosides. The two additional phosphates from the triphosphate moiety are released as by-products of the DNA synthesis reaction.
  • the process of DNA sequencing comprises a series of steps: 1) preparation of the template, 2) performing reactions to generate a series of labeled fragments that begin at a defined point and that are terminated randomly at points where one of the bases occurs in the sequence, 3) separation of the randomly-terminated fragments according to size (length in nucleotides) , 4) detection of the separated fragments, and 5) interpretation of the information contained in the pattern of separated fragments.
  • the DNA sequencing reactions that are the subject of this application are those of step 2 above.
  • Sanger dideoxynucleotide sequencing consists of annealing a short piece of synthetic DNA (called a primer) to template DNA and synthesis of new DNA by the addition of a DNA polymerase and deoxynucleosides under conditions of salts, pH, buffers and temperature that are appropriate for the DNA polymerase. Addition of dideoxynucleotides at concentrations appropriate for the DNA polymerase causes DNA synthesis to terminate. The termination occurs randomly but is a function of the ratio of the concentrations of the deoxynucleoside and the dideoxynucleotide forms of a particular base. A separate termination reaction is performed for each of the four bases.
  • Each of the four reactions results in a collection of randomly-terminated fragments and together the four collections of fragments represent the sequence information attainable from the template/primer combination.
  • DNA sequencing reactions are critical, time-consuming, labor-intensive and subject to fluctuation in efficiency from person to person.
  • a robotic system for automatically performing DNA sequencing comprising (i) a robot controlled by a programmable controller and having an arm mounted for motion in vertical and horizontal planes, (ii) first and second robot hands, said first hand being adapted to carry an microtiter plate, said second hand being adapted for pipetting, a coupling disposed on said arm for attaching each of said robot hands thereto, (iii) heating units adapted to hold a microtiter plate, (iv) a plurality of work stations, each adapted to support a microtiter plate, (v) a reagent reservoir holding unit, containing a plurality of reservoirs, and (vi) a controlled temperature storage unit.
  • Figure 1 is a plan view of the overall robotic DNA sequencing apparatus according to the current invention.
  • Figure 2 is an elevation of the robot show in Figure 1.
  • Figure 3 is an elevation, partially exploded, of the reagent reservoir shown in Figure 1.
  • Figure 4 is an elevation of two of the heater blocks shown in Figure 1.
  • Figure 5 is a longitudinal cross-section through one of the heater blocks shown in Figure 4.
  • Figure 6 is an elevation of an microtiter plate storage unit and work station.
  • Figure 7 is a plan view of a microtiter plate.
  • Figure 8 is an elevation showing the multichannel pipet suspended above a microtiter plate.
  • Figure 9 is a schematic diagram of a peristaltic pump for one of the tip washing stations shown in Figure 1.
  • Figure 10 is a flow chart of the pipet tip control method according to the current invention.
  • FIG. 1 shows an overall view of the robotic DNA sequencing system 1 of the current invention.
  • the system includes a robot 2 controlled by a controller 9.
  • the controller 9 which contains a programmable micro-processor, interfaces with a personal computer 10 that allows the operator to initiate and otherwise supervise the operation o the system. However, once initiated, the system is designed to operate automatically until all of the DNA samples have been processed.
  • Such robots are commercially available and may be purchased from the Zymark Corporation.
  • the robot 2 is comprised of an arm 5 slidably mounted for horizontal motion in a housing 29.
  • the housing 29 is slidably mounted for vertical motion on a support shaf 6 that is attached to a rotatable swivel plate 4 supported o a base 3.
  • This arrangement provides the robot 2 with a cylindrical work space defined by rotation through approximately 376°, a 35 cm range of movement in the vertica direction, and a radial extension of about 65 cm in the horizontal direction.
  • a coupling 7 is disposed at the end of the arm 7 and allows a variety of general and special purpose "hands" to be installed onto the arm 5.
  • these hands include a light duty hand 11, a heavy duty hand 12, and a specialized multichannel pipet hand 13. This last hand is shown in detail in Figure 8.
  • the system also includes six heater blocks 14, various pipet wash stations 18, a pipet tip station 15, microtiter plate work stations 16, microtiter plate storage units 17, a reagent reservoir holding unit 21, pipet storage units 19, microtiter plate controlled temperature storage units 20, and a pipet disposa station 30.
  • the reagent reservoir holding unit 21 is comprised of a cooling tank 23 to which cooled water is circulated from a cooling and pumping unit 42 via hoses 41.
  • the cooling water is maintained at approximately 4°C.
  • the tank is capable of holding and cooling eight reagent reservoirs 22.
  • a lid 24 is provided that is operated by a pneumatic operator 25 electrically controlled by the robot controller 9.
  • each heating bloc 14 has a sculptured upper surface in which ninety six trough 31 are formed.
  • the troughs have approximately the same size shape and layout as the outside surfaces of the wells 40 of plastic- standard microtiter plate 34, shown in Figures 7 and 8.
  • the heating blocks 14 have a flat support surface 32 adjacent the sculptured upper surface that is adapted to stably support the edges of a microtiter plate 34.
  • the heating blocks 14 also feature beveled edges 33 that are adapted to guide the microtiter plate onto the sculptured surface.
  • the heating blocks 14 maintain the contents of a microtiter plate 34 at approximately 65°C.
  • standard ninety six well microtiter plates 34 are utilized, with each well having a capacity of about 300 ⁇ l.
  • the wells 40 are arranged in twelve columns, with eight wells in each column. In the preferred embodiment, only the first seven columns Cl to C7 are utilized.
  • each storage unit 17 is comprised of a number of compartments 36, each of which is adapted to hold a single microtiter plate 34 with its lid 35.
  • the microtiter plate work stations 16 comprise a flat work surface 35 adapted to support an microtiter plate 34.
  • pipet wash stations 18 are disposed adjacent the heating blocks 14 and microtiter plate work stations 16.
  • the pipet storage units 19 are each comprised of a vertical array of storage compartments, similar to the microtiter plate storage unit 17, that are adapted to hold boxes of plastic disposable pipet tips 50 prior to their insertion onto the multichannel pipet hand 13, as shown in Figure 8.
  • the temperature controlled storage units 20 have doors 26 that are electrically operated by the robot controller 9.
  • the temperature controlled storage units 20 have a vertical array of storage compartments each of which is adapted to hold two pairs of microtiter plates, each pair consisting of one microtiter plates stacked on another. The two pairs are stored next to one another.
  • the storage units 20 are maintained at a temperature of 6°C by the use of circulating cooled water.
  • the storage units 20 may be equipped with a vacuum forming unit if it is desired to dry the reagents.
  • Sequenase available from US Biochemicals
  • Taq DNA polymerase available form Perkins-Elmer Cetus
  • Bst DNA polymerase available from Bio-Rad
  • Reaction conditions are based on the two-step reaction in which the initial extension of the primer and incorporation of radiolabeled nucleotide is performed as a separate step from the termination step.
  • Radiolabeled dATP 35 S-dATP; 33 P-dATP
  • solutions of deoxynucleosides and dideoxynucleotides available from Boehringer Mannheim Biochemicals
  • Optimal ratios of dideoxynucleotide to deoxynucleoside may be determined empirically.
  • Products of sequencing reactions are separated on 6% DNA sequencing gels (Sequagel 6%; National Diagnostics) that are fixed in 10% methanol-10% acetic acid and dried under vacuum, and exposed to X-ray film to produce autoradiographs.
  • the system allows thirty micro-titer plates 34 and lids 35 to be stored at roo temperature prior to sequencing and a maximum of forty plate with completed sequencing reactions to be stored in the cooled storage units 20. Therefore, the robot 2 can perform a maximum of 240 sequencing reactions (30 plates, 8 reaction per plate) in a single run.
  • the system can, however, be configured to allow ambient, as well as cooled storage, of a maximum of sixty microtiter plates and, therefore, will allo a maximum of 480 reactions to be performed in a single unattended run.
  • Sequencing reactions as performed manually, impose significant constraints on automation.
  • the chief constraint is to pipet, reproducibly and precisely, volumes as small as 0.5 ⁇ l.
  • pipetting, reproducibly and precisely, a volume of 0.5 ⁇ l is beyond the capacity of most automated pipetting systems.
  • One solution to this problem involves the use of dried reagents since they allow the use of larger volumes.
  • the dried reagents in the target wells occupy no volume, therefore, the volume that is transferred can be larger by the volume that is normally occupied by the dried reagents.
  • Use of dried reagents allow the use of volumes that for the smallest volumes are in the range of seven microliters, volumes that can be pipetted reproducibly and precisely by many automated systems.
  • the invention may be practiced by performing sequencing reactions using reagents dried under vacuum onto microtiter plates.
  • the system would include cooled storage units 20 that could be placed under vacuum to dry down the reagents on microtiter plates.
  • Such a system must: operate without intervention after the initial setup, have cooled storage that incorporated a robotically controlled lid for reagents, have cooled storage that could be placed under vacuum for microtiter plates, have several (preferably six or more) heat blocks sculpted to fit microtiter plates, be capable of manipulating microtiter plates and be capable of reproducibly pipetting volumes as small as seven microliters from microtiter plate wells containing as little as twelve microliters.
  • the problems of pipetting small volumes may also be overcome by the use of surfactants along with a novel method of controlling the movement of the multichannel pipet hand 13 into the microtiter plate wells 40, thereby allowing the use of reagent solutions.
  • the robot controls the movement of the multichannel pipet hand 13 so that the pipet tips 50 touch the sides of the wells 40 while dispensing, so as to wipe the tips. Touching the sides provided a surface for the drops to run down, thereby preventing the solution from beading back onto the tip surface, since such beading would prevent all of the volume aspirated from being dispensed. Wiping of the tips resulted in increased reproducibility of the dispensed volume.
  • the operating system for the robot controller 9 utilizes an interpreted language with macro-like modules that can be nested to seven layers.
  • Standard programming provided by the robot supplier may be used to control some basic functions that involve the coordination of power-and-event controller switches, such as the opening and closing of doors 26 of the temperature controlled storage units 20.
  • software necessary to adequately control the robot's ability to pipet small volumes of about 7 ⁇ l volumes or less from source volumes of about 12 ⁇ l has not heretofore been available.
  • the multichannel micropipet hand 13 itself is capable of pipetting small volumes reproducibly and precisely if the pipet tips 50 are properly located within the well, the dexterity of the robot 2 in moving the multichannel micropipet hand 13 into the well 40 is crucial to accuracy.
  • Figure 8 shows the multichannel micropipet han 13 suspended above a microtiter plate 34 just prior to insertion of the tips 50 into the wells 40 for aspiration.
  • the inventors have found that accurate pipetting of small volumes from small source volumes requires that the pipet tips 50 reach to just above the bottoms of the microtiter plate wells 40. If the tips 50 are too high, they will not be immersed in the liquid for the last few microliters. If the tips 50 touched the bottoms of the wells with excessive force, however, the holes of the tips will be blocked and the recovery variable.
  • the robot is equipped with force sensors 60, which may be of the strain gage type, that are coupled to th arm 5, as shown in Figure 2.
  • the sensors 60 provide a measure of the force exerted on the arm in arbitrary units.
  • the feedback from the sensors 60 enables the arm 5 with the micropipet hand 13 to sense when the tips 50 make contact with the bottoms of the wells 40. After the tips 50 make contact, the height of the arm 5 is increased slightly to prevent the blockage of the tips.
  • the force sensors 60 lack precision and report variable readings depending on the recent movements of the arm 5. Specifically, the inventors have found that the value obtained from the force sensor 60 with the hand 13 is hanging freely is higher when the hand had recently been moved upward than when it had recently been moved downward. Also the sensitivity of the sensor 60 is insufficient to detect contact by an absolute measure.
  • a novel pipet tip movement control method a flow chart for which is shown in Figure 10, for sensing when the tips 50 are at the appropriate location in the bottom of the wells 40 of a microtiter plate 34 for proper aspiration.
  • This location is that location in which the pipet tips 50 almost touch the microtiter plate 34 so that the tips are not pressed against the bottoms of the wells, which would imped flow into them, nor are they so far above the bottoms of the wells as to prevent complete aspiration.
  • the inventors have found that when the contact point is achieved, the microtiter plate can not be easily lifted from the sculptured surface of a heat block 14 but that the tips, which are somewhat flexible, do not show any signs of being subjected to force, such as bowing out. In additional, it has been found that when the contact point is achieved, it is possible to fractionally lift the microtiter plate off the heating block 14 if the height of the hand 13 were increased by 0.2 mm.
  • the sensing of bottoms of the wells is refined by moving the pipet tips 50 toward the bottoms of the wells in a series of increasing smaller decrements in the height of the pipet tip above the wells 40, accomplished by a logic module referred to as "bottom sensing" in Figure 10. Since the force sensed by the sensor 60 includes a force component attributable to the weight of the freely hanging multichannel pipet hand 13, the measured force will decrease when the pipet tips 50 contact the bottoms of the well because the wells will then be supporting at least a portion of the weight of the hand. Therefore, each incremental decrease in height is alternated with a decision step that tests whether the force on the pipet tips, as measured by the sensor 60, is sufficiently small to indicate that the tips have contacted the bottoms of the wells.
  • the force on the pipet tips 50 be sufficiently great to bow the tips outward in order to be assured that contact with the bottoms of the wells has occurred. Moreover, the point at which the tips are bowed is achieved incrementally to provide reproduciblity. Thus, the value that the measured force must be below, to satisfy the criteria for "bottom sensing" according to the method, is set so that at such a value of measured force, the pipet tips 50 can be expected to be bowed outward.
  • reliable aspiration can not be achieved with the pipet tips pressing against the well bottoms with such force since their inlets are apt to be blocked by the contact.
  • the contact point sensing module lifts the arm in very small increments, the first of which is equal to the product of a small constant k times the number of loops, TRYA, through the bottom sensing module that were necessary to satisfy the "bottom sensing" condition. This incrementing is continued until the detected force is equal to the force sensed when the hand 13 was hanging freely, thereby indicating that the pipet tips are not pressing into the wells such that flow into them may be impeded.
  • the constant ⁇ f2 is equal to the difference in the force measured with a freely hanging hand between a measurement taken after the hand was moved up and a measured taken after the hand was moved down and need be established only once to account for the peculiarities of the particular robot system.
  • the bottom sensing module is attempted from the beginning one more time (route B in Figure 10) . If they fail to touch the bottoms of the wells after an additional four passes through the bottom sensing module, the robot is halted. Once the height necessary to achieve the "contact point" is determined, its value is stored and reused each time the hand 13 returns to that particular microtiter plate to aspirate another column of wells. Thus, the point of contact is determined only once for each set of eight sequencing reactions performed on a given microtiter plate.
  • contact point sensing is determined for each microtiter plate when the column of wells containing the extended primer and template DNA together with the other reagents including the DNA polymerase are aspirated just prior to dispensing the reaction to the four termination mixes.
  • the multichannel pipet control method of the current invention may be explained as follows.
  • step 100 the values of the counters TRYA and TRYB are zeroed after the hand 13 has moved into position above the microtiter plate as shown in Figure 8.
  • the force sensor is standardized by always moving toward and traversing the microtiter plate 34 in a similar manner — e.g., moving radially toward and across the plate — prior to beginning the method.
  • the force F as sensed by the force sensor 60 which is equal to the force from a freely hanging hand after the hand has been moved downward, is measured and stored as F re in step 101.
  • the force F from the sensor 60 is measured again and it is determined whether or not this force is less than F ref - ⁇ f ⁇ , ⁇ fi being an empirically determined constant based on the particular robot, thereby indicating that bottom sensing has occurred.
  • step 104 the height H is incremented downward by a second increment h- , ⁇ h-
  • step 105 the force F from the sensor 60 is measured again and it is again determined whether or not this force is less than F ref - ⁇ f ⁇ . If it is less, the contact point sensing module is executed. If it is not, then in step 106 the height H is incremented down by a increment ⁇ h 2 , ⁇ h 2 being less than ⁇ h-
  • step 107 the force F from the sensor 60 is measured again and it is determined whether or not this forc is less than F ref - ⁇ f.*
  • step 111 TRYB is increased by one and TRYA set to zero.
  • step 112 the height is increased by the amount of the original decrement ⁇ h 0 so that further execution of the bottom sensing module is begun with a height that is less than the original height by 4x( ⁇ h 1 + ⁇ h 2 + ⁇ h 3 ) .
  • step 113 it is determined whether or not TRYB is less than two. If it is, then steps 101 to 113 are repeated. If it is not, then the operation is halted and the problem investigated.
  • the contact point sensing module is initiated in step 115 by raising the height H of the arm by H-l-(TRYA x k) , k being an empirically determined constant based on the particular robot.
  • H-l-(TRYA x k) k being an empirically determined constant based on the particular robot.
  • the amount by which the height of the hand 13 is raised that is, the increase in height necessary to ensure that the tips 50 are not pressing agains the wells with sufficient force to impede flow yet to also ensure that the tips are not moved so far from the bottom of the well that aspiration will be incomplete — is a function of the number of times the group of incrementing steps 104 t 108 were performed during the execution of the bottom sensin module that attained the contact point.
  • step 116 the force F is measured again and it is determined if it is greater than F ref - ⁇ f 2 , ⁇ f 2 being an empirically determined constant to correct for the history effect, as previously discussed. If it not, then the height H is raised again by the increment ⁇ h 3 and steps 11 to 119 are repeated. If it is greater, then the value of H re is set to H so that repetitions of the aspiration on the same microtiter plate can be repeated using the same height value determined above during the first aspiration on a particular microtiter plate. Lastly, in step 119 the aspiration of the contents of the wells 40 in the particular microtiter plate column are aspirated.
  • Empirically determined constants for height differences are about as follows: ⁇ h 0 is slightly less than the height difference measured between the bottoms of the micropipet tips and the bottoms of the microtiter plate wells when the micropipet tip hand is in the position suspended over the wells. It is dependent on the definitions for the positions at and above the heating blocks 14, taught to the robot.
  • the starting value is about equal to ⁇ f 2 as defined above. All empirical values are refined by trials.
  • tip washing stations 18 are disposed adjacent those positions where the robot performs pipetting operations.
  • plastic inserts are used similar to those used for the cooled reagent reservoir 22, except that the tip washing plastic inserts have a nominal volume of 38 ml, whereas the cooled reagent reservoir plastic insert have a nominal volume of 18 ml.
  • the tip washing stations 18 are mounted such that they are radially in line with the microtiter plate and their upper edge is at the sam height as the upper edge of a microtiter plate at the particular pipetting station, as shown in Figures 4 and 6.
  • a system driven by a peristaltic pumps 61 and 62 may be used to replenish the water in the tip washing stations 18 so as to maintain the volume at a constant level and prevent the accumulation of reagents during long runs may also be incorporated into the system.
  • th system in addition to the pumps 61 and 62, th system includes a source reservoir 63 and a waste reservoir 64, each in flow communication with a tip washing station 18 by means of hoses 65 and 66.
  • Tips are washed by aspirating and expelling 50 ⁇ l several times. To ensure that little or no water is transferred to the next reaction, with a resulting dilution of the reagents, the last expulsion of the washing solution is performed slowly while the tips are touching the side of and upper edge of the tip washing station to prevent beading of the water back onto the tips.
  • Another problem encountered when the microtiter plate format is used to facilitate automation of DNA sequencing according to the current invention is that evaporation of reagents occurs during the various heating steps in the reactions. This problem is solved in the current invention by the addition of non-ionic detergents containing water to compensate for volume loss due to evaporation, as explained further below.
  • the system can perform 240 reactions in a single unattended run of seven hours.
  • the robot configuration can be altered to allow 480 reactions to be performed in an unattended run of fourteen hours. Therefore if a daytime run of 240 reactions in seven hours is combined with a nighttime run of 480 reactions in fourteen hours, a total of 720 reactions could be performed per day.
  • the process of DNA sequencing consists of a series of steps: 1) preparation of the template, 2) performing reactions to generate a series of labeled, base-specific, randomly terminated fragments, 3) separation of the randomly-terminated fragments according to length, 4) detection of the separated fragments, and 5) interpretation of the information contained in the pattern of separated fragments. Automation of the steps results in greater throughput of sequences. Automation of all of the steps is essential for improved throughput in a single laboratory. Automation of the reactions necessary to generate the series of fragments is discussed in detail below.
  • preparation of DNA template can be performed with instruments such as the Biomek 1000 work station (available from Beckman) or Autogen 540 (available from Autogen) .
  • Detection of the separated fragments and interpretation of the information can be performed with fluorescence-based sequence detection systems coupled to computers such as the AB1-370 (available for Applied Biosystems) , the A.L.F. (available from Pharmacia) , or autoradiograph-based computer graphics systems such as that supplied by Bio-Rad or the Bio Image (available from Millipore) scanners and analysis systems.
  • computers such as the AB1-370 (available for Applied Biosystems) , the A.L.F. (available from Pharmacia) , or autoradiograph-based computer graphics systems such as that supplied by Bio-Rad or the Bio Image (available from Millipore) scanners and analysis systems.
  • many computer software packages are available.
  • the DNA sequencing method according to the current invention is begun by manually loading the reservoirs 22 with reagents. Enzyme plus label is placed in reservoir no. 1, terminations G, A, T and C, respectively, in reservoirs nos. 2-5, the STOP buffer in reservoir no. 6 and water in reservoir no. 7. Boxes of pipet tips 50 are manually loaded into the pipet storage units 19 and the DNA sample is manually deposited into the wells 40 of up to three microtiter plates 34. Because the samples are used eight at a time, one column at a time, and there are twelve columns on a microtiter plate 34, and because the maximum number of samples that can be processed in the preferred embodiment is 240 samples, up to three microtiter plates 34 containing samples may be placed on work surfaces of the robot. Two of the three microtiter plates may have all ninety six wells forty completely filled with samples and the third may have up to forty eight wells 40, wells in column 1 through column 6, filled.
  • more than one microtiter plate is used to increase the number of reactions performed in a set time. This is accomplished by programming the robot to perform two sets of reactions that are out of phase by five minutes, therefore, samples are deposited into the sample microtiter plates so that there are always even numbers of columns of samples.
  • the reaction conditions for Bst DNA polymerase are modified such that each of the basic steps (i.e., heating or denaturation, annealing, labeling/extension and termination) are performed for about equal lengths of time — i.e., in blocks of about five minutes.
  • the robot 2 steps through the sequence of operations programmed into the controller 9, performing the reactions on a pair of samples for each cycle, as explained above, and performing additional cycles until all of the samples have been sequenced.
  • the light weight hand 11 is used to move boxes of micropipet tips 50 from the micropipet tip storage units 19 and to move microtiter plates 34 from microtiter plate storage units 17.
  • the heavy duty hand is used to manipulate microtiter plates 34 and lids 35, when they are on the heating blocks 14 and especially when the microtiter plates 34 with lids 35 are stacked on one another and moved to the temperature controlled storage units 20.
  • the robot 2 using the light weight hand 11 removes a box of tips 50 from the tip storage 19 and places it on the tip work station 15. All of the sample plates are first placed in the temperature controlled storage unit 20 using the heavy duty hand 12. The first microtiter plate 34 containing samples is then placed on a work station 16. Next, using the light duty hand 11, two empty microtiter plates 34 are removed from a microtiter plate storage unit 17 and placed on each of two of the heater blocks 14 in the outer row of heating blocks and the lids 35 of the microtiter plates are removed and placed on an adjacent work station 16 or a heating block 14. Note that the system 1 incorporates an outer row of heating blocks 14, in addition to an inner row, so that heating can be carried out at two temperatures.
  • heating is only necessary at one temperature so that the outer row of heating blocks 14 function merely as work stations and do not produce heat.
  • the lid of the microtiter plate containing samples on work station 16 is removed and placed on an adjacent work station 16.
  • the robot aspirates one column of wells 40 from the sample containing microtiter plate on the work station 16 and dispenses them into the first column of one of the empty microtiter plates on the outer row of heating blocks.
  • the tips 50 are then washed in the tip washing station 18. These steps are repeated so that the first column of the second, previously empty, microtiter plate on the outer row of heating blocks is also filled with sample.
  • the lids of the microtiter plates on the heating blocks are replaced and one of the microtiter plates from the outer row of heating blocks is placed on an active heating block 14 in the first row, whereupon it is heated for five minutes at 65°C to complete the denaturation step.
  • the lid of the microtiter plate that contained samples is replaced and the microtiter plate is then returned from work station 16 to the storage unit 20 if there are still samples remaining on the microtiter plate. If no samples are remaining, the microtiter plate is disposed of to free up the work station 16.
  • the other microtiter plate from the outer row of heating blocks is placed in the storage unit 20.
  • microtiter plate containing the now denatured sample is then placed on a work station 16 where it is held at room temperature for five minutes to complete the annealing step. Its lid is removed and placed on an adjacent work station 16.
  • the reagents are aspirated from the reservoirs 22 and dispensed into the microtiter plate containing the denatured sample.
  • the STOP buffer is dispensed into column no. 6, followed by tip washing in the tip washing station 18.
  • the water is then aspirated and dispensed from its reservoir 22 into column no. 7 of the microtiter plate and the tips are again washed.
  • the terminations G, A, T, and C are then aspirated from their reservoirs 22 and dispensed into column nos. 2-5 of the microtiter plate, with the tips being washed between each operation.
  • the tips 50 are disposed of in the tip disposal unit 30 and a new set of tips from the tip work station 15 are placed on the multichannel pipet 13.
  • the enzyme/label is aspirated from its reservoir 22 directly into the DNA sample in column no. 1 of the microtiter plate.
  • the lid of the microtiter plate is replaced and the microtiter plate is then placed on one of the active heating blocks 14 to begin the five minute label and extension step while being heated to 65 ⁇ C.
  • the lid of the microtiter plate is removed to an adjacent heating block 14 and the reaction volume in column 1 is supplemented with the water from column 7 by aspirating water from column 7 and dispensing into column 1.
  • the five minute termination step is then begun by sequentially pipetting portions of sample from column no.
  • This microtiter plate is then stacked on top of the other microtiter plate in a pair, which by now has also been processed in the sequential, out of phase method previously discussed, and both are stored in the storage unit 20 while the second pair of samples are processed. Processing and storing of successive pairs of samples is continued until all of the samples are sequenced.
  • the micropipet tips 50 in a box in the tip station 15 are depleted the empty box is disposed of by returning it to a rack in a tip storage 19. A new box of micropipet tips 50 is then moved from the tip storage 19 to the tip station 19.
  • the samples are placed on a sequencing gel the gels are electrophoresed, fixed, dried an X-rayed film is exposed to it to generate an autoradiogram t identify the DNA sequence using techniques well known in the art.
  • Sequenase is an enzyme that has become a standard for sequencing in many laboratories. To compare the sequencing reactions performed by the robot with those performed routinely in other laboratories, the reaction conditions for Sequenase were modified for use on the robot. DNA and primer were heated to 65 °C for five min. , the primer was annealed to the DNA at room temperature for four min. , the enzyme-labeling mix was added and the reaction incubated for four minutes at room temperature, the extension-reaction products were dispensed into the termination mixes and the reactions were incubated at 37 °C for five min. Sequencing reactions were stopped by the addition of STOP buffer. Because of the constraints on pipetting, the volumes of the reactions had to be increased.
  • the annealing volume was increased to 16 ⁇ l as compared with 10 ⁇ l in the standard protocol. After annealing the effective volume was about 10 ⁇ l.
  • the buffer in the annealing mix was adjusted t be correct for the effective volume.
  • the enzyme-labeling mi was essentially identical to the standard protocol except that non-ionic detergents were added to a final concentratio of 0.02% and the volume of enzyme dilution buffer was about one-third of the standard protocol.
  • the volume was increased by the addition of 9 ⁇ l of water to about 25 ⁇ l.
  • Five microliters of the labeling-extension reaction was dispensed to each of the 5 ⁇ l termination mixes.
  • the reactio was terminated by the addition of four ⁇ l STOP buffer.
  • sequences generated were comparable to those generated by an individual experienced at sequencing. Furthermore, there was little if any variation from well to well, and from plate to plate for a single template.
  • Sequenase is, however, labile.
  • the manufacturer recommends not storing the diluted enzyme for more than 60 min. at 4 °C.
  • To achieve the goal of long periods of unattended operation it was essential to use an enzyme that was stable in solution at 4 °C at the working dilution.
  • the DNA polymerase from Bacillus stearother ophilus (Bst DNA polymerase) is reported to be extremely stable and is functional for sequencing even after fourteen days of storage at room temperature. Therefore, we investigated its use for the robot.
  • the sequencing conditions were similar to those developed for the use of Sequenase with the robot except that all steps were performed for about five minutes and the elongation-labeling and termination steps were performed at 65 °C.
  • Bst DNA polymerase gave excellent results and was stable in the reagent reservoir for at least 24 hours.
  • the system according to the current invention could also be used in a diagnostic DNA sequencing laboratory, wher speed, quality, and reproducibility are of paramount importance. Analysis of the sequences of the same gene or cDNA fragments from different patients makes it possible to produce rapidly large numbers of DNA sequencing templates especially if the PCR is employed.
  • the demonstrated low-volume pipetting dexterity could be employed for sequencing with non-radioactive labels, as well. Use of fluorescent tags would accommodate those DNA sequencing projects or facilities that utilize automated detection systems. Accordingly, the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

Abstract

L'invention se rapporte à un système robotique grâce auquel on effectue des réactions de séquençage d'acide nucléique dans une structure de plaques de microtitrage. Le robot (2) est composé d'un bras (5) et de son unité de commande (9), de mains (11, 12, 13) avec lesquelles il peut manipuler des plaques de microtitrage avec couvercles et des plaques de microtitrage sans couvercles avec lesquelles il peut effectuer un prélèvement à la pipette dans une plage de 5 à 200 microlitres; de chambres (20) de stockage de refroidissement dont les portes (26) peuvent être commandées électroniquement pour stocker les plaques de microtitrage, d'une cuve de refroidissement (23) avec couvercle à commande électronique pour stocker les réactifs de séquençage de l'acide nucléique; de blocs chauffants (14) pour incuber les réactions à des températures appropriées dépassant la température ambiante; d'une unité de stockage (15) pour boîtes de pointes cônes de micropipettes; d'une unité de stockage (17) pour plaques de microtitrage dans lesquelles seront effectuées les réactions de séquençage et un logiciel pour commander le robot lors de prélèvements à la pipette.
PCT/US1993/009961 1992-10-16 1993-10-15 Procede et appareil d'execution robotique de reactions de sequencage de didesoxynucleotides de sanger WO1994008759A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997022882A1 (fr) * 1995-12-18 1997-06-26 Alan Norman Hale Appareil et procede de traitement d'echantillons
WO1997044670A1 (fr) * 1996-05-21 1997-11-27 Abion Beteiligungs- Und Verwaltungs-Verwaltungs-Gesellschaft Mbh Appareil automatique de pipettage
WO1998040159A2 (fr) * 1997-03-10 1998-09-17 Trega Biosciences, Inc. Procede et dispositif de synthese chimique combinatoire
FR2760844A1 (fr) * 1997-03-17 1998-09-18 Genethon Ii Dispositif et procede d'analyse automatique d'echantillons sur gels
WO2000033962A1 (fr) * 1998-12-07 2000-06-15 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Appareil de thermocyclage rotatif
WO2000058736A1 (fr) * 1999-03-25 2000-10-05 Coulter International Corp. Dispositif d'aspiration de liquide dans un recipient
DE10122913A1 (de) * 2001-05-11 2003-02-06 Urban Liebel Vorrichtung und Verfahren zum Bearbeiten biotechnologischer Proben
WO2004108270A2 (fr) * 2003-06-05 2004-12-16 Bioprocessors Corp. Systeme et procede destines a une automatisation
US7008788B2 (en) * 2001-07-30 2006-03-07 Agilent Technologies, Inc. Containers for supports comprising biopolymers
WO2008081199A2 (fr) * 2007-01-06 2008-07-10 Preptec Solutions Limited Appareil et méthode de nettoyage de surfaces
WO2009059022A1 (fr) 2007-10-30 2009-05-07 Complete Genomics, Inc. Appareil pour le séquençage rapide d'acides nucléiques
EP2082061A2 (fr) * 2006-10-06 2009-07-29 Vandalia Research, Inc. Procédé pour un système à cycle thermique rapide et continu
US9080208B2 (en) 2010-07-22 2015-07-14 Gencell Biosystems Ltd. Composite liquid cells
WO2015120398A1 (fr) * 2014-02-10 2015-08-13 Gencell Biosystems Limited Dispositif de préparation de banque d'acides nucléiques médiée par des cellules à liquides composites (clc), et ses procédés d'utilisation
US9776182B2 (en) 2012-11-27 2017-10-03 Gencell Biosystems Ltd. Handling liquid samples

Families Citing this family (79)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6943034B1 (en) 1991-11-22 2005-09-13 Affymetrix, Inc. Combinatorial strategies for polymer synthesis
EP0916396B1 (fr) * 1991-11-22 2005-04-13 Affymetrix, Inc. (a Delaware Corporation) Stratégies associées pour la synthèse de polymères
US7625697B2 (en) 1994-06-17 2009-12-01 The Board Of Trustees Of The Leland Stanford Junior University Methods for constructing subarrays and subarrays made thereby
JP3307816B2 (ja) * 1995-12-19 2002-07-24 シスメックス株式会社 試料吸引装置
US7244622B2 (en) * 1996-04-03 2007-07-17 Applera Corporation Device and method for multiple analyte detection
JP2001505648A (ja) * 1996-07-05 2001-04-24 ベックマン コールター インコーポレイテッド 自動試料処理システム
WO1998018549A1 (fr) * 1996-10-30 1998-05-07 Sumitomo Chemical Co., Ltd. Systeme d'automatisation des experiences de synthese, appareil de traitement et de separation de liquides et cuve de reaction
US6008010A (en) * 1996-11-01 1999-12-28 University Of Pittsburgh Method and apparatus for holding cells
US5779984A (en) * 1996-12-04 1998-07-14 Rainin Intstrumental Co., Inc. Pipette tip rack and refill pack containing large maximized volume freely nestable pipette tips
US5985214A (en) * 1997-05-16 1999-11-16 Aurora Biosciences Corporation Systems and methods for rapidly identifying useful chemicals in liquid samples
DE59811623D1 (de) * 1997-08-08 2004-08-05 Aventis Pharma Gmbh Pipettierroboter mit Temperiervorrichtung
WO1999014368A2 (fr) * 1997-09-15 1999-03-25 Whitehead Institute For Biomedical Research Procede et appareil pour le traitement d'un echantillon d'analyte biomoleculaire au moyen d'un dispositif microfabrique
US6323035B1 (en) * 1997-09-24 2001-11-27 Glaxo Wellcome, Inc. Systems and methods for handling and manipulating multi-well plates
US6803019B1 (en) 1997-10-15 2004-10-12 Aclara Biosciences, Inc. Laminate microstructure device and method for making same
FR2771179B1 (fr) * 1997-11-14 1999-12-31 Inst Francais Du Petrole Dispositif de manipulation d'objets de tres petites dimensions
US6893877B2 (en) 1998-01-12 2005-05-17 Massachusetts Institute Of Technology Methods for screening substances in a microwell array
US6627446B1 (en) * 1998-07-02 2003-09-30 Amersham Biosciences (Sv) Corp Robotic microchannel bioanalytical instrument
US6103199A (en) * 1998-09-15 2000-08-15 Aclara Biosciences, Inc. Capillary electroflow apparatus and method
CA2346268A1 (fr) * 1998-10-16 2000-04-27 Intelligent Automation Systems Poste de travail automatique de traitement continu
US6443022B1 (en) 1999-01-14 2002-09-03 Intelligent Automation Systems, Inc. Fluid level detector and method for use with automated workstation
NZ513390A (en) 1999-03-19 2003-01-31 Genencor Int Multi-through hole testing plate for high throughput screening
US20020151040A1 (en) 2000-02-18 2002-10-17 Matthew O' Keefe Apparatus and methods for parallel processing of microvolume liquid reactions
US6719949B1 (en) * 2000-06-29 2004-04-13 Applera Corporation Apparatus and method for transporting sample well trays
WO2002014868A1 (fr) * 2000-08-16 2002-02-21 Council Of Scientific And Industrial Research Procede rapide de dosage immunoenzymatique facilite par l'utilisation de micro-ondes
US20030055571A1 (en) * 2000-12-15 2003-03-20 Olympus Optical Co., Ltd. Novel computation with nucleic acid molecules, computer and software for computing
US20030059823A1 (en) * 2001-09-21 2003-03-27 Juki Corporation Hybridization apparatus and method for detecting nucleic acid in sample using the same
CN1653338A (zh) 2002-05-17 2005-08-10 贝克顿·迪金森公司 用于分离、放大和检测目标核酸序列的自动化系统
US8277753B2 (en) 2002-08-23 2012-10-02 Life Technologies Corporation Microfluidic transfer pin
EP1608952B1 (fr) 2002-12-20 2016-08-10 Life Technologies Corporation Appareil et procede de dosage utilisant des reseaux microfluidiques
WO2004099787A1 (fr) * 2003-05-05 2004-11-18 Visible Diagnostics A/S Systeme de transport automatique d'echantillons et utilisation de ce systeme
JP4351875B2 (ja) * 2003-07-15 2009-10-28 シスメックス株式会社 液体吸引装置とそれを備えた分析装置
US20050186578A1 (en) * 2004-02-20 2005-08-25 Sven Bulow Chamber array arrangement
AU2005222618A1 (en) 2004-03-12 2005-09-29 Biotrove, Inc. Nanoliter array loading
US20100140173A1 (en) * 2004-12-03 2010-06-10 Chris Suh Method and Device for Gravity Flow Chromatography
US20090223893A1 (en) * 2005-12-01 2009-09-10 Gjerde Douglas T Method and Device for Desalting an Analyte
US20100170852A1 (en) * 2004-12-03 2010-07-08 Chris Suh Method and Device for Gravity Flow Chromatography
JP5897780B2 (ja) 2005-01-28 2016-03-30 デューク ユニバーシティ プリント回路基板上の液滴操作装置及び方法
WO2007024778A2 (fr) * 2005-08-22 2007-03-01 Applera Corporation Dispositif, systeme et procede destines a deposer des volumes distincts de fluide immiscible traite
JP4328787B2 (ja) * 2005-10-04 2009-09-09 キヤノン株式会社 核酸試料検査装置
CN103254896B (zh) * 2006-03-21 2015-01-21 超点公司 在可见区或近红外区中发光的发光材料
US9476856B2 (en) 2006-04-13 2016-10-25 Advanced Liquid Logic, Inc. Droplet-based affinity assays
US8492168B2 (en) * 2006-04-18 2013-07-23 Advanced Liquid Logic Inc. Droplet-based affinity assays
US20140193807A1 (en) 2006-04-18 2014-07-10 Advanced Liquid Logic, Inc. Bead manipulation techniques
US10078078B2 (en) 2006-04-18 2018-09-18 Advanced Liquid Logic, Inc. Bead incubation and washing on a droplet actuator
US7439014B2 (en) 2006-04-18 2008-10-21 Advanced Liquid Logic, Inc. Droplet-based surface modification and washing
US7851184B2 (en) * 2006-04-18 2010-12-14 Advanced Liquid Logic, Inc. Droplet-based nucleic acid amplification method and apparatus
US8809068B2 (en) 2006-04-18 2014-08-19 Advanced Liquid Logic, Inc. Manipulation of beads in droplets and methods for manipulating droplets
US7901947B2 (en) 2006-04-18 2011-03-08 Advanced Liquid Logic, Inc. Droplet-based particle sorting
WO2009111769A2 (fr) 2008-03-07 2009-09-11 Advanced Liquid Logic, Inc. Réactif et préparation et chargement d’un échantillon sur un dispositif fluidique
US9046514B2 (en) 2007-02-09 2015-06-02 Advanced Liquid Logic, Inc. Droplet actuator devices and methods employing magnetic beads
US9116129B2 (en) 2007-05-08 2015-08-25 Idexx Laboratories, Inc. Chemical analyzer
WO2009032863A2 (fr) 2007-09-04 2009-03-12 Advanced Liquid Logic, Inc. Actionneur de gouttelette avec substrat supérieur amélioré
KR20100100974A (ko) 2007-12-23 2010-09-15 어드밴스드 리퀴드 로직, 아이엔씨. 액적 작업들을 수행하는 액적 작동기 구성부 및 방법
US8852952B2 (en) 2008-05-03 2014-10-07 Advanced Liquid Logic, Inc. Method of loading a droplet actuator
US8926065B2 (en) 2009-08-14 2015-01-06 Advanced Liquid Logic, Inc. Droplet actuator devices and methods
US9091649B2 (en) 2009-11-06 2015-07-28 Advanced Liquid Logic, Inc. Integrated droplet actuator for gel; electrophoresis and molecular analysis
EP2322130B1 (fr) * 2009-11-16 2013-01-16 TRUMPF Medizin Systeme GmbH + Co. KG Unité d'alimentation médicale
EP2510073B1 (fr) 2009-12-08 2017-07-05 Omnipv, Inc. Matériaux luminescents émettant de la lumière dans le domaine visible ou dans l'infrarouge proche, et procédés de formation associés
EP2516669B1 (fr) 2009-12-21 2016-10-12 Advanced Liquid Logic, Inc. Analyses d'enzymes sur un diffuseur à gouttelettes
EP2520369A1 (fr) * 2011-05-05 2012-11-07 Eppendorf AG Procédé et appareil de laboratoire permettant de traiter les échantillons de laboratoire
CA2833897C (fr) 2011-05-09 2020-05-19 Advanced Liquid Logic, Inc. Retroaction microfluidique utilisant une detection d'impedance
JP5714410B2 (ja) * 2011-05-16 2015-05-07 株式会社日立ハイテクノロジーズ 自動分析装置及び方法
JP5703377B2 (ja) * 2011-06-24 2015-04-15 株式会社日立ハイテクノロジーズ 核酸増幅装置及び核酸分析装置
BR112014000257A2 (pt) 2011-07-06 2017-03-01 Advanced Liquid Logic Inc armazenamento de reagente em um atuador de gota
US9513253B2 (en) 2011-07-11 2016-12-06 Advanced Liquid Logic, Inc. Droplet actuators and techniques for droplet-based enzymatic assays
WO2013016413A2 (fr) 2011-07-25 2013-01-31 Advanced Liquid Logic Inc Dispositif et système d'actionneur à gouttelettes
US20140170735A1 (en) * 2011-09-25 2014-06-19 Elizabeth A. Holmes Systems and methods for multi-analysis
US9664702B2 (en) 2011-09-25 2017-05-30 Theranos, Inc. Fluid handling apparatus and configurations
US9632102B2 (en) 2011-09-25 2017-04-25 Theranos, Inc. Systems and methods for multi-purpose analysis
US8475739B2 (en) 2011-09-25 2013-07-02 Theranos, Inc. Systems and methods for fluid handling
US9810704B2 (en) 2013-02-18 2017-11-07 Theranos, Inc. Systems and methods for multi-analysis
WO2013078216A1 (fr) 2011-11-21 2013-05-30 Advanced Liquid Logic Inc Dosages de la glucose-6-phosphate déshydrogénase
IN2015DN00359A (fr) 2012-06-27 2015-06-12 Advanced Liquid Logic Inc
US11545241B1 (en) 2013-09-07 2023-01-03 Labrador Diagnostics Llc Systems and methods for analyte testing and data management
US9797916B2 (en) 2014-01-10 2017-10-24 Idexx Laboratories, Inc. Chemical analyzer
JP6255058B2 (ja) * 2016-05-16 2017-12-27 株式会社タカゾノテクノロジー 水剤供給装置
FR3062133B1 (fr) 2017-01-23 2022-06-17 Interscience Procede et appareil de comptage de colonies
LU500344B1 (en) * 2021-06-29 2022-12-29 Dispendix Gmbh Tool for determining a position of a dispensing head
EP4184176A1 (fr) * 2021-11-17 2023-05-24 Roche Diagnostics GmbH Procédé de détection d'un fond d'au moins un puits

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4715413A (en) * 1985-10-18 1987-12-29 Backlund Ulf E H H Apparatus for manipulating small volumes of liquid
US4984475A (en) * 1989-07-24 1991-01-15 Tritech Partners Ultra low carryover sample liquid analysis apparatus and method
WO1991016675A1 (fr) * 1990-04-06 1991-10-31 Applied Biosystems, Inc. Laboratoire de biologie moleculaire automatise

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CH548597A (de) * 1972-06-14 1974-04-30 Greiner Electronic Ag Verfahren zum absaugen einer fluessigkeit und vorrichtung zur durchfuehrung des verfahrens.
US4483382A (en) * 1983-09-14 1984-11-20 The Goodyear Tire & Rubber Company Tire bead with layer of cellular elastic material beneath bead core
US4794085A (en) * 1984-07-19 1988-12-27 Eastman Kodak Company Apparatus and method for detecting liquid penetration by a container used for aspirating and dispensing the liquid
JPH0833320B2 (ja) * 1986-03-20 1996-03-29 株式会社東芝 自動化学分析装置
DE3614955C1 (de) * 1986-05-02 1987-08-06 Schulz Peter Proben-Verteiler-System
GB8716279D0 (en) * 1987-07-10 1987-08-19 Amersham Int Plc Performing nucleic acid reactions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4715413A (en) * 1985-10-18 1987-12-29 Backlund Ulf E H H Apparatus for manipulating small volumes of liquid
US4984475A (en) * 1989-07-24 1991-01-15 Tritech Partners Ultra low carryover sample liquid analysis apparatus and method
WO1991016675A1 (fr) * 1990-04-06 1991-10-31 Applied Biosystems, Inc. Laboratoire de biologie moleculaire automatise

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ADVANCES IN LABORATORY AUTOMATION ROBOTICS, Zymark Corporation, issued 1987, S. HAMILTON, "Robotic Assays for Fermentation Products", pages 1-21. *

Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997022882A1 (fr) * 1995-12-18 1997-06-26 Alan Norman Hale Appareil et procede de traitement d'echantillons
WO1997044670A1 (fr) * 1996-05-21 1997-11-27 Abion Beteiligungs- Und Verwaltungs-Verwaltungs-Gesellschaft Mbh Appareil automatique de pipettage
WO1998040159A2 (fr) * 1997-03-10 1998-09-17 Trega Biosciences, Inc. Procede et dispositif de synthese chimique combinatoire
WO1998040159A3 (fr) * 1997-03-10 1999-01-07 Trega Biosciences Inc Procede et dispositif de synthese chimique combinatoire
US6045755A (en) * 1997-03-10 2000-04-04 Trega Biosciences,, Inc. Apparatus and method for combinatorial chemistry synthesis
FR2760844A1 (fr) * 1997-03-17 1998-09-18 Genethon Ii Dispositif et procede d'analyse automatique d'echantillons sur gels
WO1998041874A1 (fr) * 1997-03-17 1998-09-24 Genethon Ii Dispositif et procede d'analyse automatique d'echantillons sur gels
US6448066B1 (en) 1998-12-07 2002-09-10 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Rotary thermocycling apparatus
WO2000033962A1 (fr) * 1998-12-07 2000-06-15 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Appareil de thermocyclage rotatif
WO2000058736A1 (fr) * 1999-03-25 2000-10-05 Coulter International Corp. Dispositif d'aspiration de liquide dans un recipient
DE10122913A1 (de) * 2001-05-11 2003-02-06 Urban Liebel Vorrichtung und Verfahren zum Bearbeiten biotechnologischer Proben
US7008788B2 (en) * 2001-07-30 2006-03-07 Agilent Technologies, Inc. Containers for supports comprising biopolymers
WO2004108270A2 (fr) * 2003-06-05 2004-12-16 Bioprocessors Corp. Systeme et procede destines a une automatisation
WO2004108270A3 (fr) * 2003-06-05 2005-04-21 Bioprocessors Corp Systeme et procede destines a une automatisation
EP2082061A2 (fr) * 2006-10-06 2009-07-29 Vandalia Research, Inc. Procédé pour un système à cycle thermique rapide et continu
EP3527671A1 (fr) * 2006-10-06 2019-08-21 Applied DNA Sciences Inc. Appareil pour un système à cycle thermique rapide en continu
EP2082061A4 (fr) * 2006-10-06 2013-01-16 Vandalia Res Inc Procédé pour un système à cycle thermique rapide et continu
WO2008081199A2 (fr) * 2007-01-06 2008-07-10 Preptec Solutions Limited Appareil et méthode de nettoyage de surfaces
WO2008081199A3 (fr) * 2007-01-06 2008-11-20 Preptec Solutions Ltd Appareil et méthode de nettoyage de surfaces
EP2215209A1 (fr) * 2007-10-30 2010-08-11 Complete Genomics, Inc. Appareil pour le séquençage rapide d'acides nucléiques
EP2215209A4 (fr) * 2007-10-30 2012-08-29 Complete Genomics Inc Appareil pour le séquençage rapide d'acides nucléiques
WO2009059022A1 (fr) 2007-10-30 2009-05-07 Complete Genomics, Inc. Appareil pour le séquençage rapide d'acides nucléiques
US10017815B2 (en) 2007-10-30 2018-07-10 Complete Genomics, Inc. Method for high throughput screening of nucleic acids
US9382585B2 (en) 2007-10-30 2016-07-05 Complete Genomics, Inc. Apparatus for high throughput sequencing of nucleic acids
US9493816B2 (en) 2010-07-22 2016-11-15 GenCell Biosytems Ltd. Composite liquid cells
US10125389B2 (en) 2010-07-22 2018-11-13 Gencell Biosystems Limited Composite liquid cells
US9080208B2 (en) 2010-07-22 2015-07-14 Gencell Biosystems Ltd. Composite liquid cells
US9776182B2 (en) 2012-11-27 2017-10-03 Gencell Biosystems Ltd. Handling liquid samples
US10252261B2 (en) 2012-11-27 2019-04-09 Gencell Biosystems Ltd. Handling liquid samples
WO2015120398A1 (fr) * 2014-02-10 2015-08-13 Gencell Biosystems Limited Dispositif de préparation de banque d'acides nucléiques médiée par des cellules à liquides composites (clc), et ses procédés d'utilisation
US10384187B2 (en) 2014-02-10 2019-08-20 Gencell Biosystems Ltd Composite liquid cell (CLC) mediated nucleic acid library preparation device, and methods for using the same

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